Methods of inhibiting osteoclast activity

ABSTRACT

Methods of inhibiting osteoclastogenesis and the activity of osteoclasts are disclosed. Methods of treating patients who have diseases characterized bone loss are disclosed. According to the methods, an amount of a TRANCE/RANK inhibitor effective to inhibit osteoclastogenesis is administered to the patient. Pharmaceutical compositions which comprise TRANCE/RANK inhibitor in an amount effective to inhibit osteoclastogenesis. Methods of modulating dendritic cell maturation, T cell proliferation, and/or CD40 receptor systems in an individual are disclosed. The methods comprise the step of administering to the individual an amount of a TRANCE/RANK inhibitor effective to modulating dendritic cell maturation, T cell proliferation, and/or CD40 receptor systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to application Ser. No.60/146,094 filed Jul. 28, 1999, which is incorporated herein byreference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT RIGHTS

The present invention was made under Grant EY09332 from the NationalInstitutes of Health. The Government may have certain rights to theinvention.

FIELD OF THE INVENTION

The present invention relates to the methods of down-modulatingosteoclastogenesis activity, thereby inhibiting bone matrix erosion andthus preventing bone loss and treating bone diseases.

BACKGROUND OF THE INVENTION

Osteoclasts are large multinuclear cells which function to erode bonematrix. They are related to macrophage and other cells that develop frommonocyte cells. Like macrophage, osteoclasts are derived fromhaematopoietic progenitor cells.

Bone matrix erosion is a normal process which occurs in coordinationwith bone matrix formation, a process in which osteoblasts are involved.Essentially, osteoclasts erode bone matrix and tunnel into bone whileosteoblasts follow, line the walls of the tunnel and form new bonematrix. Typically, in a normal adult, about 5-10% of bone is replaced bythese processes annually.

Bone diseases such as osteoporosis and Paget's disease are characterizedby a loss of bone. Similarly, metastatic bone disease, rheumatoidarthritis and peridontal bone disease are also characterized by boneloss. In many cases, bone loss leads to fractures in patients. Inaddition to the pain and suffering, patients become physically impairedwhich often leads to complications having negative consequences onpatient health and quality of life. Moreover, the economic costsattributable to these diseases are tremendous.

Receptors and ligands of the Tumor Necrosis Factor family have recentlybeen shown to play an essential part in the differentiation and activityof osteoclasts. On the one hand, Tumor Necrosis Factor-α (TNF-α) isknown to promote osteoclastogenesis. On the other hand, a TNF-likemolecule present on and/or secreted by osteoclasts and stromal cells,referred to interchangeably in the field and herein as Receptoractivator of NF-κB ligand, (RANKL), Osteoclast differentiation factor(ODF), Osteoprotegerin ligand (OPGL), and TNF-related activation-inducedcytokine (TRANCE), interacts with a TNF-receptor-like molecule, referredto in the field and herein as Receptor activator of NF-κB ligand,(RANK), which present in the membranes of osteoclast precursors andmature osteoclasts to regulate osteoclastogenesis and the resorbingactivity of mature osteoclasts. The utilization of TNF-α antagonists,such as a monoclonal antibodies, for therapeutic purposes, has provendifficult, however, because of immunity to the large molecule, andlimited entry into some specialized compartments of the body. Suda, etal. 1999 Endocrine Reviews 20(3):345-357, which is incorporated hereinby reference, describes osteoclast differentiation and function.Filvaroff, E and R. Derynck 1998 Cutr. Biol. 8:R679-R682, which isincorporated herein by reference, refer to bone remodeling and asignaling system for osteoclast regulation.

There is a need for methods of regulating osteoclastogenesis and theresorbing activity of mature osteoclasts. There is a need for methods ofpreventing bone loss and treating bone diseases.

SUMMARY OF THE INVENTION

The present invention relates to methods of inhibitingosteoclastogenesis and the resorbing activity of mature osteoclasts.According to the present invention, an amount of a TRANCE/RANK inhibitoreffective to inhibit osteoclast bone erosion activity is administered toa patient.

The present invention relates to methods of treating patients who havediseases characterized bone loss. According to the present invention, anamount of a TRANCE/RANK inhibitor effective to inhibitosteoclastogenesis is administered to the patient.

The present invention relates to pharmaceutical compositions whichcomprise a TRANCE/RANK inhibitor in an amount effective to inhibitosteoclastogenesis.

The present invention relates to methods of modulating dendritic cellmaturation, T cell proliferation, and/or CD40 receptor systems in anindividual comprising the step of administering to the individual anamount of a TRANCE/RANK inhibitor effective to modulating dendritic cellmaturation, T cell proliferation, and/or CD40 receptor systems.

The present invention relates to peptides that inhibitosteoclastogenesis, modulating dendritic cell maturation, T cellproliferation, and/or CD40 receptor systems

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, the term “TRANCE/RANK inhibitors” refers to thecompounds which inhibit osteoclastogenesis and/or osteoclast functionand which are described in PCT application Ser. No. PCT/US99/15062 filedJul. 1, 1999 and entitled “CAVITY INDUCED ALLOSTERIC MODIFICATION OFINTERMOLECULAR INTERACTIONS AND METHODS OF IDENTIFYING COMPOUNDS THATEFFECT THE SAME”, which is incorporated herein by reference. TRANCE/RANKinhibitors can function as an antagonist of the cellular receptor RANKby inhibiting TRANCE/RANK. The Example below describes an assay whichcan be performed to identify peptides that inhibit osteoclastogensisand/or osteoclast function.

As used herein, the term “diseases characterized by bone loss” is meantto refer to diseases, conditions, disorders and syndromes which have asa symptom or pathology a decrease in bone mass or density. Examples ofdiseases characterized by bone loss include osteoporosis, Paget'sdisease, metastatic bone disease, rheumatoid arthritis and peridontalbone disease.

As used herein, the term “therapeutically effective amount” is meant torefer to an amount of a compound which produces a medicinal effectobserved as reduction in the rate of bone loss in an individual when atherapeutically effective amount of a compound is administered to anindividual who is susceptible to or suffering from a diseasecharacterized by bone loss. Therapeutically effective amounts aretypically determined by the effect they have compared to the effectobserved when a composition which includes no active ingredient isadministered to a similarly situated individual.

The invention provides methods for treating individuals that havediseases characterized by bone loss. TRANCE/RANK inhibitors areadministered to the individual in an amount effective to inhibitosteoclastogenesis and/or osteoclast function and thereby reduce boneloss, i.e. a therapeutically effective amount.

The invention also provides novel therapeutic pharmaceuticalcompositions for treating diseases characterized by bone loss. Thepharmaceutical compositions comprise a therapeutically effective amountof TRANCE/RANK inhibitors and a pharmaceutically acceptable carrier ordiluent. In preferred embodiments, the pharmaceutical compositions areinjectable pharmaceutical compositions, i.e. they are sterile,pyrogen-free, free of particulate matter, essentially isotonic with andotherwise suitable for injection into humans.

Applicants have discovered that the compounds and peptides describedherein are useful to inhibit osteoclastogenesis and/or osteoclastfunction. By inhibiting osteoclastogenesis and/or osteoclast function,bone erosion can be prevented and bone loss can be reduced. Patientssuffering from diseases characterized by bone loss can be treated byadministering an amount of compounds effective to inhibitosteoclastogenesis and/or osteoclast function. In addition, patientsidentified as being susceptible to diseases characterized by bone losscan be prophylactically treated by administering an amount of compoundseffective to inhibit osteoclastogenesis and/or osteoclast function.

Individuals who have a disease characterized by bone loss can beidentified by those having ordinary skill in the art by well knowndiagnostic means and criteria. Individuals who are susceptible to adisease characterized by bone loss can be identified by those havingordinary skill in the art based upon family medical history and/or thepresence of genetic markers or genes associated with a diseasecharacterized by bone loss.

According to some embodiments of the invention, TRANCE/RANK inhibitorsuseful in the invention are compounds having Formula I which is setforth in the section below entitled Formulae. In compounds of Formula I,R₁ and R₂ are, independently, selected from the group consisting of —H,—OCH₃, —CH₂CH₃, -t-butyl, 3-carboxy-4-chlorophenylamino, —N—(CH₂CH₂OH)₂,and —O(O)C—Ph. R₃ is selected from the group consisting of —H, ethyl,—OCH₃, Cl, Br, F, 3-carboxy-4-chlorophenylamino, —N—(CH₂CH₂OH)₂,-t-butyl, and —OC(O)—Ph, and is not limited to attachment at any certainposition on the phenyl ring to which it is attached. Preferably, R₃ isattached at either the 1 or 4 position of the phenyl ring. R₄ isselected from the group consisting of —Br,—Cl, and —F.

In some preferred compounds of Formula I:

R₁, R₂, and R₃ are —OCH₃, R₃ is attached at the 4 position, and R₄ is—Cl;

R₁ and R₂ are methyl, R₃ is ethyl, attached at the 4 position, and R₄ is—Cl;

R₁ and R₂ are —OCH₃, R₃ is —Cl, attached at the 2 position, and R₄ is—Cl;

R₁ and R₂ are —OCH₃ and R₃ is H, and R₄ is —Cl;

R₁ is H, R₂ and R₃ are 3-carboxy-4-chlorophenylamino, and R₃ is attachedat the 4 position, and R₄ is —Cl;

R₁ and R₂ are —N(CH₂CH₂OH)₂, R₃ is Cl, attached at the 4 position, andR₄ is —Cl;

R₁, R₂, and R₃ are t-butyl, R₃ is attached at the 4 position, and R₄ is—Cl;

R₁ is —OCH₃, R₂and R₃are H, and R₄ is Cl; or

R₁, R₂, and R₃ are benzoate, R₃ is attached at the 4 position, and R₄ is—Br.

Some preferred compounds of Formula I have the structures I-A, I-B, I-C,I-D, I-E, I-F, I-G, I-H or I-I which are set forth below in the sectionentitled Formulae.

These compounds are available from the following suppliers:

Compound Catalog Number Supplier I-A F36,700-1 Aldrich, Milwaukee, WII-B S11,245-3 Aldrich, Milwaukee, WI I-C 00569 Ryan Scientific, Isle ofPalms, S.C. I-D F10,001-3 Aldrich, Milwaukee, WI I-E 00129 George UHE,Paramus, NJ I-F F37,166-1 Aldrich, Milwaukee, WI I-G S-11,239-9 Aldrich,Milwaukee, WI I-H F-27,721-5 Aldrich, Milwaukee, WI I-I F12,920-8Aldrich, Milwaukee, WI

In some embodiments, the TRANCE/RANK inhibitors are compounds havingFormula II which is set forth below in the section entitled Formulae. Incompounds having Formula II, R₁ is selected from the group consisting of-diphenylchloro methyl, -di(4-chlorophenyl)chloro methyl, and 4-(diphenylchloromethyl)phenyl; and R₂, R₃, R₄ are independently selectedfrom the group consisting of —Br, —Cl, and —F, and are preferably —Cl.

Preferred compounds of Formula II have the structures II-A, II-B, II-Cand II-D which are set forth below in the section entitled Formulae.These compounds are available from the following suppliers:

Compound Catalog Number Supplier II-A S5,479-9 Aldrich, Milwaukee, WlII-B S5,755-0 Aldrich, Milwaukee, WI II-C S5,740-2 Aldrich, Milwaukee,WI II-D S5,751-8 Aldrich, Milwaukee, WI

According to some embodiments of the invention, TRANCE/RANK inhibitorsuseful in the invention are compounds having Formula III which is setforth in the section below entitled Formulae. In compounds of FormulaIII, R can be any of the following:

R₁═(NO₂), O(CO)CH₃, OH, O(CO)CH₃, O(CO)(CH₂)₂COOH, O(CO)CH₂Br,O(CO)CH₂Cl, O(CO)CH₂N(CH₃)₃, or OC₅H₉O;

R₂═CH₂O(NO₂), CHO, CH₂O(NO₂), CN, CH₃, COOH, CHNOH, CH₂O(CO)(CH₂)₂COOH,CHN(NH)CONH₂, CHN(NH)C₆H₅, CHN(CH₂)C₆H₅, CH₂N(CH₂)₂OH, CH₂NC₆H₅, orCH₂N(NH)CSNH₂;

R₃═OH, or H;

R₄═CH₃;

R₅═OH;

R₆═C₄H₃O₂, N(NHCO)C₆H₄Cl, N(NHCO)C₆H₄F, COOH, O, COCH₃,CH(CH₃)(CH₂)₂COOH, CH(CH₃)(CH₂)₂COOCH₃, O(CO)C₆H₅, or OH;

R₇═O(CO)CH₂N(CH₃)₃, or O(CO)CH₃;

R₈═OH;

R₉═O, or OH;

R₁₀═O.

Some preferred compounds of Formula III have the structures III-1through III-31 which are set forth below in the section entitledFormulae and are described in the table below which includes cataloguenumbers from ChemDiv, Inc., San Diego, Calif.

Number Catalogue # Formula III-1 0449-0070 R1=O(NO₂); R2=CH₂O(NO₂);R3=R5=OH; R4= CH₃; R6=C₄H₃O₂ III-2 0449-0037 R1=R3=R5=R8=OH;R2=CHO;R4=CH₃; R6= C₄H₃O₂ III-3 0449-0071 R1=R3=R5=OH; R2=CH₂O(NO2);R6=C₄H₃O₂ III-4 0449-0077 R1=O(CO)CH₃; R2=CN; #=R5=OH; R4=CH₃; R6=C₄H₃O₂III-5 0449-0095 R1=R3=R5=OH; R2=R4=CH₃; R6=C₄H₃O₂ III-6 0449-0101R1=R3=R5=OH; R2=COOH;R4=CH₃; R6= C₄H₃O₂ III-7 0449-0112R1=O(CO)CH₃;R2=CHO; R3=R5=OH;R4=CH₃;R6=C₄H₃O₂ III-8 0449-0113R1=O(CO)CH3; R2=R4=CH3; R3=H;R5=OH; R6=C₄H₃O₂ III-9 0449-0115R1=O(CO)(CH₂)₂COOH; R2=CHO; R3=R5=OH; R4=CH₃; R6=C₄H₃O₂ III-10 0449-0116R1=R3=R5=OH;R2=CHNOH; R4=CH₃; R6= C₄H₃O₂ III-11 0449-0119R1=O(CO)(CH₂)_(2COOH; R2=) CH₂O(CO)(CH₂)₂COOH; R3=R5=OH; R4=CH₃;R6=C₄H₃O₂ III-12 0449-0120 R1=O(CO)CH₂Br; R2=CH₃; R3=H;R4=CH₃; R5=OH;R6=C₄H₃O₂ III-13 0449-0160 R1=O(CO)CH₂Cl; R2=CHO;R3=R5=OH; R4=CH₃;R6=C₄H₃O₂ III-14 0449-0719 R1=OH;R2=R4=CH₃; R6=N(NHCO)C₆H₄Cl III-150449-0720 R1=OH;R2=R4=CH₃; R6=N(NHCO)C₆H₄F III-16 N001-0005 R1=R5=OH;R2=CHO; R3=H;R4=CH₃; R6=C₄H₃O₂ III-17 N008-0012 R1=O(CO)CH₂N(CH₃)₃;R2=R4=CH₃;R5=OH; R6=C₄H₃O_(2;) R7=O(CO)CH₂N(CH₃)₃ III-18 N023-0001R1=OH;R2=R4=CH₃; R3=R5=OH;R6=C₄H₃O_(2;) R7=O(CO)CH₃ III-19 N023-0004R1=OH;R2=CHN(NH)CONH₂; R3=R5=OH; R4=CH₃; R6=C₄H₃O₂ III-20 N023-0005R1=R3=R5=OH;R2=CHN(NH)C₆H₅; R4=CH3;R6=C₄H₃O₂ III-21 N023-0006R1=R3=R5=OH;R2=CHN(CH₂)C₆H₅; R4=CH3 ;R6=C₄H₃O₂ III-22 N023-0007R1=OH;R2=CH₂N(CH₂)₂OH; R4=CH₃; R3=R5=OH; R6=C₄H₃O₂ III-23 N023-0008R1=OH;R2=CH₂NC₆H₅; R3=R5=OH; R4=CH₃; R6=C₄H₃O₂ III-24 N023-0025R1=OH;R2=CH₂N(NH)CSNH₂;R2=R5=OH; R4=CH₃;R6=C₄H₃O₂ III-25 N039-0025R1=OH; R2=R4=CH₃; R3=H; R6=COCH₃ III-26 S003-0002 R1=OH; R2=R4=CH₃;R3=H; R6=COOH III-27 S003-0006 R1=OH; R2=R4=CH₃; R3=H;R6=CH(CH₃)(CH₂)₂COOH; R9=O III-28 S003-0007 R1=OH; R2=R4=CH₃; R3=H;R6=CH(CH₃)(CH₂)₂COOCH₃; R9=OH; R10=O III-29 S003-0009 R2=R4=CH₃;R3=H;R6=O III-30 S003-0014 R1=OH; R2=R4=CH₃;R3=H;R6=O(CO)C₆H₅ III-31S003-0012 R1=O(C₅H₉O); R2=R4=CH₃;R3=H;R6=OH

According to some embodiments of the invention, TRANCE/RANK inhibitorsuseful in the invention are compounds having Formula IV which is setforth in the section below entitled Formulae. In compounds of FormulaIV, R can be any of the following:

R₁═O(CO)(CH₂)₂COOH, or O(CO)CH₂Br;

R₂═O(CO)(CH₂)₂COOH, or O(CO)CH₂Br.

Some preferred compounds of Formula IV have the structures IV-1 throughIV-2 which are set forth below in the section entitled Formulae and aredescribed in the table below which includes catalogue numbers fromChemDiv, Inc., San Diego, Calif.

Number Catalogue # Formula IV-1 0521-0013 R1=R2=O(CO)(CH₂)₂COOH IV-20521-0014 R1=R2=O(CO)CH₂Br

According to some embodiments of the invention, TRANCE/RANK inhibitorsuseful in the invention are compounds having Formula V which is setforth in the section below entitled Formulae. In compounds of Formula V,R can be any of the following:

R₁═O, OH, or O(CO)CH₃

R₂═O(CO)CH₃, OH, CO(CH₃), or CO(CH₂)O(CO)CH₃,

R₃═CH₃, or OH

R₄ ═O(CO)CH₂C₆H₄I, or CH₃

Some preferred compounds of Formula V have the structures V-1 throughV-5 which are set forth below in the section entitled Formulae and aredescribed in the table below which includes catalogue numbers fromChemDiv, Inc., San Diego, Calif.

Number Catalogue # Formula V-1 N017-0002 R1=O; R2=O(CO)CH₃; R3=CH₃ V-2N017-0003 R1=OH; R2=OH; R3=CH₃ V-3 N017-0005 R1=O(CO)CH₃; R2=CO(CH₃);R3=OH; R4=O(CO)CH₂C₆H₄I V-4 N017-0006 R1=O; R2=CO(CH₂)O(CO)CH₃; R3=OHV-5 N017-0012 R1=OH; R2=CO(CH₃); R3=OH; R4=CH₃

According to some embodiments of the invention, TRANCE/RANK inhibitorsuseful in the invention are compounds having Formula VI which is setforth in the section below entitled Formulae. In compounds of FormulaVI, R can be any of the following:

R₁═O(CO)CH₃, OH, or O(CO)(CH₂)₂COOH

R₂ CH₃

R₃═O, or OH

R₄═CH₃;

R₅═C₉H₁₃COCH₃, C₉H₁₃(CH₂CH₃)(CH₂OH), C₉H₁₃(CH₂CH₃)(CH₂OCOCH₃),C₉H₁₃(CH₂CH₃)(CH₂OCO(CH₂)₂COOH), C₉H₁₃(CH₂CH₃)(COOH), orC₈H₇O(CH₃)(C₄H₉OCH₃)

R₆═CH₃

R₇═O, or H

R₈═CH₃

R₉═(CH₃)₂

R₁₀═Br.

Some preferred compounds of Formula V have the structures VI-1 throughVI-11 which are set forth below in the section entitled Formulae and aredescribed in the table below which includes catalogue numbers fromChemDiv, Inc., San Diego, Calif.

Number Catalogue # Formula VI-1 N017-0018 R1=O(CO)CH₃; R2=CH₃;R3=O;R7=H;R4=CH₃; R5=C9H₁₃COCH₃ VI-2 N017-0019 R1=OH; R2=R4=CH₃;R3=OH;R5=C₉H₁₃COCH₃ VI-3 N032-0001 R1=OH; R9=(CH₃)₂; R2=R6=R8=CH₃;R5=C₉H₁₃(CH₂CH₃)(CH₂OH) VI-4 N032-0002 R1=O(CO)CH3; R9=(CH₃)₂;R2=R6=R8=CH₃; R5=C₉H₁₃(CH₂CH₃)(CH₂OH) VI-5 N032-0003 R1=OH;R9=(CH₃)_(2; R2=R6=R8=CH) ₃; R5=C₉H₁₃(CH₂CH₃)(CH₂O(CO)CH₃) VI-6N032-0004 R1=O(CO)(CH₂)₂COOH; R9=(CH₃)₂; R2=R6=R8=CH₃;R5=C₉H₁₃(CH₂CH₃)CH₂O(CO)(CH₂)₂COOH VI-7 N032-0006 R1=O; R2=R6=R8=CH₃;R5=C₉H₁₃(CH₂CH₃) (COOH); R9=(CH₃)₂ VI-8 N039-0023 R1=O(CO)CH₃;R2=R4=CH₃; R5= C₈H₇O(CH₃)C₄H₉OCH₃) VI-9 N039-0029 R1=OCOCH₃; R2=R4=CH₃;R3=OH; R7=O; R10=Br; R5=C₈H₇O(CH₃)C₄H₉OCH₃) VI-10 N039-0031 R1=OH;R2=R4=CH3; R3=OH; R7=O; R5=C₈H₇O(CH₃)C₄H₉OCH₃) VI-11 N039-0032 R1=OH;R2=R4=CH3; R7=OH; R5=C₈H₇O(CH₃)C₄H₉OCH₃)

According to some embodiments of the invention, TRANCE/RANK inhibitorsuseful in the invention are compounds have structures of Formulae VII toXII set forth in the section below entitled Formulae. Compounds ofFormulae VIII to XII and are described in the table below which includescatalogue numbers from ChemDiv, Inc., San Diego, Calif.

Number Catalogue # VII 0836-0110 VIII N002-0041 IX N039-0046 X S003-0004XI S003-0018 XII N001-0001

According to the invention, TRANCE/RANK inhibitors useful in theinvention to treat diseases characterized by bone loss may be formulatedand administered as follows. The compounds of the invention, may beadministered to a subject per se or in the form of a pharmaceuticalcomposition. Pharmaceutical compositions comprising the compounds of theinvention may be manufactured by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the active peptides or peptide analoguesinto preparations which can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

For topical administration the compounds of the invention may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Alternatively, the compounds may be in powder form for constitution witha suitable vehicle, e.g., sterile pyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be readily formulated bycombining the active peptides or peptide analogues with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thecompounds of the invention to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a patient to be treated. For oral solid formulationssuch as, for example, powders, capsules and tablets, suitable excipientsinclude fillers such as sugars, such as lactose, sucrose, mannitol andsorbitol; cellulose preparations such as maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP); granulating agents; and binding agents. Ifdesired, disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

If desired, solid dosage forms may be sugar-coated or enteric-coatedusing standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

For buccal administration, the compounds may take the form of tablets,lozenges, etc. formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat may be used to deliver peptides and peptide analogues of theinvention. Certain organic solvents such as dimethylsulfoxide also maybe employed, although usually at the cost of greater toxicity.Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

As the compounds of the invention may contain charged side chains ortermin, they may be included in any of the above-described formulationsas the free acids or bases or as pharmaceutically acceptable salts.Pharmaceutically acceptable salts are those salts which substantiallyretain the antimicrobial activity of the free bases and which areprepared by reaction with inorganic acids. Pharmaceutical salts tend tobe more soluble in aqueous and other protic solvents than are thecorresponding free base forms.

The compounds of the invention will generally be used in an amounteffective to achieve the intended purpose. For use to treat or preventTNF-associated disorders, the compounds of the invention, orpharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. By therapeutically effective amount ismeant an amount effective ameliorate or prevent the symptoms, or prolongthe survival of, the patient being treated. Determination of atherapeutically effective amount is well within the capabilities ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture (i.e., theconcentration of test compound that inhibits 50% of TRANCE/RANK-bindinginteractions). Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the compounds which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5to 1 mg/kg/day. Therapeutically effective serum levels may be achievedby administering multiple doses each day. Some preferred dosages rangefrom 1 nM to 500 mM. Some preferred dosages range from 1 mM to 500 mM.Some preferred dosages range from 1 mg to 500 mg. Some preferred dosagesrange from 1000 mg to 3000 mg. Some preferred dosages range from 1500 mgto 2500 mg. According to the invention, TRANCE/RANK inhibitors areadministered one to four times per day.

In cases of local administration or selective uptake, the effectivelocal concentration of the compounds may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

The amount of compound administered will, of course, be dependent on thesubject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The therapy may be repeated intermittently while symptoms detectable oreven when they are not detectable. The therapy may be provided alone orin combination with other drugs.

Preferably, a therapeutically effective dose of the compounds describedherein will provide therapeutic benefit without causing substantialtoxicity.

Toxicity of the compounds described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Compoundswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a dosage range that is not toxic for use human. The dosageof the compounds described lies preferably within a range of circulatingconcentrations that include the effective dose with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fing1 et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1,p.1). Preferred dosages range from 1 nM to 500 mM.

Pharmaceutical compositions according to the present invention compriseTRANCE/RANK inhibitors formulated as described above in therapeuticallyeffective doses. In some embodiments, the pharmaceutical compositions issterile and pyrogen free.

Other aspects of the present invention include the use of TRANCE/RANKinhibitors in methods involving other cell types in which TRANCE/RANKmediated signaling is involved in cell development and/or activity. Suchcell types include antigen presenting cells such as dendritic cells andlymphocytes. Anderson et al. 1997 Nature 390:175-179, refer to theRANK/RANKL in T cells and dendritic cells. Similarly, Kong et al. 1999Immunol. and Cell Biology 77:188-193 refer to osteoprotegerin ligand asa common link between osteoclastogenesis, lymphnode formation andlymphocyte development. In addition, Wong et al. 1999 J. LeukocyteBiology 65:715-724 refer to TRANCE as regulating dendritic cell andosteoclast function. TRANCE/RANK inhibitors formulated as describedabove in effective doses can be used to modulate dendritic cellmaturation and function, T cell proliferation and CD40 receptor systems.

According to some aspects of the invention, the present inventionrelates to novel peptides and methods using such novel peptides.

The aromatically modified peptides of the invention comprise an aminoacid sequence that consists of 27 amino acid residues and has theformula:

R₁—R₂—R₃—R₄—R₅

wherein:

R₁ is 1-5 amino acid residues;

R₂ is a linking amino acid residue;

R₃ is selected from the group consisting of: DRGWA (SEQ ID NO:1); DGDLAT(SEQ ID NO:2); SDFATE (SEQ ID NO:3); VTKTSIKIPSSH (SEQ ID NO:4);TKTSIKIPSSH (SEQ ID NO:5); KTSIKIPSSH (SEQ ID NO:6); YWSNSEF (SEQ IDNO:7); YWNSE (SEQ ID NO:8); PDQDAP (SEQ ID NO:9); PDSWH (SEQ ID NO:10);SKEL (SEQ ID NO:11); EIEF (SEQ ID NO:12); SRSGHS (SEQ ID NO:13);RFQEEIKENTKNDKQ (SEQ ID NO:14); TSYPD (SEQ ID NO:15); KENTK (SEQ IDNO:16); and conservatively substituted derivatives thereof;

R₄ is a linking amino acid residue;

R₅ is 1-5 amino acid residues; and

wherein R₂ and R₄ are bound to each other, thereby forming a cyclicportion which includes R₂, R₃ and R₄ with R₁ and R₅ forming exocyclicportions, and one or both of R₁ and R₅ comprising at least one tyrosineor phenylalanine.

The aromatically modified peptides of the invention comprise an aminoacid sequence that consists of 27 amino acid residues and has theformula:

R₁—R₂—R₃—R₄—R₅

wherein:

R₁ is 1-5 amino acid residues including at least one tyrosine orphenylalanine;

R₂ is cysteine;

R₃ is selected from the group consisting of: DRGWA (SEQ ID NO:1); DGDLAT(SEQ ID NO:2); SDFATE (SEQ ID NO:3); VTKTSIKIPSSH (SEQ ID NO:4);TKTSIKIPSSH (SEQ ID NO:5); KTSIKIPSSH (SEQ ID NO:6); YWSNSEF (SEQ IDNO:7); YWNSE (SEQ ID NO:8); PDQDAP (SEQ ID NO:9); PDSWH (SEQ ID NO:10);SKEL (SEQ ID NO:11); EIEF (SEQ ID NO:12); SRSGHS (SEQ ID NO:13);RFQEEIKENTKNDKQ (SEQ ID NO:14); TSYPD (SEQ ID NO:15); KENTK (SEQ IDNO:16); and conservatively substituted derivatives thereof;

R₄ is cysteine;

R₅ is 1-5 amino acid residues including at least one tyrosine orphenylalanine; and

wherein R₂ and R₄ are bound to each other, thereby forming a cyclicportion which includes R₂, R₃ and R₄ with R₁ and R₅ forming exocyclicportions.

The aromatically modified peptides of the invention comprise an aminoacid sequence that consists of 27 amino acid residues and has theformula:

R₁—R₂—R₃—R₄—R₅

wherein:

R₁ is selected from the group consisting of: absent, Y; RYQEE (SEQ IDNO:17); and conservatively substituted derivatives thereof;

R₂ is cysteine;

R₃ is selected from the group consisting of: DRGWA (SEQ ID NO:1); DGDLAT(SEQ ID NO:2); SDFATE (SEQ ID NO:3); VTKTSIKIPSSH (SEQ ID NO:4);TKTSIKIPSSH (SEQ ID NO:5); KTSIKPSSH (SEQ ID NO:6); YWSNSEF (SEQ IDNO:7); YWNSE (SEQ ID NO:8); PDQDAP (SEQ ID NO:9); PDSWH (SEQ ID NO:10);SKEL (SEQ ID NO:11); EIEF (SEQ ID NO:12); SRSGHS (SEQ ID NO:13);RFQEEIKENTKNDKQ (SEQ ID NO:14); TSYPD (SEQ ID NO:15); KENTK (SEQ IDNO:16); and conservatively substituted derivatives thereof;

R₄ is cysteine;

R₅ is selected from the group consisting of Y; Y-[NH2]; YDE; YDE-[NH2];YVKQE (SEQ ID NO:18); YVKQE-[NH2]; YKHR (SEQ ID NO:19); YKHR-[NH2]; I;I-[NH2]; DKQ and CDKQ-[NH2] and conservatively substituted derivativesthereof; and wherein R₂ and R₄ are bound to each other, thereby forminga cyclic portion which includes R₂, R₃ and R₄ with R₁ and R₅ formingexocyclic portions, and one or both of R₁ and R₅ comprising at least onetyrosine or phenylalanine.

In preferred embodiments, the peptide is selected from the groupconsisting of: YCDRGWACY (SEQ ID NO:20); YCDGDLATCY (SEQ ID NO:21);YCSDFATECY (SEQ ID NO:22); YCVTKTSIKIPSSHCY (SEQ ID NO:23);YCKTSIKIPSSHCY (SEQ ID NO:24); YCYWSNSEFCY (SEQ ID NO:25); CYWNSECY (SEQID NO:26); YCPDQDAPCY (SEQ ID NO:27); YCPDSWHCYDE (SEQ ID NO:28);YCSKELCYVKQE (SEQ ID NO:29); YCEIEFCYKHR (SEQ ID NO:30); YCSRSGHSCY (SEQID NO:31); YCRFQEEIKENTKNDKQCY (SEQ ID NO:32); YCTSYPDCI (SEQ ID NO:33);RYQEECKENTKCDKQ (SEQ ID NO:34); and conservatively substitutedderivatives thereof.

Preferred peptides according to the invention are:

SEQ ID NOs:20-30 with amidated C termini

RL 1-1: [H]-YC DRGWA CY-[NH2]

RL 2-1: [H]-YC DGDLAT CY-[NH2]

RL 2-2: [H]-YC SDFATE CY-[NH2]

RL 3-1: [H]-YC VTKTSIKIPSSH CY-[NH2]

RL 3-2: [H]-YC KTSIKIPSSH CY-[NH2]

RL 4-1: [H]-YC YWSNSEF CY-[NH2]

RL 4-2: [H]-C YWNSE CY-[NH2]

RL 5-1: [H]-YC PDQDAP CY-[NH2]

OP 1: [H]-YC PDSWH CYDE-[NH2]

OP 2: [H]-YC SKEL CYVKQE-[NH2]

OP 3: [H]-YC EIEF CYKHR-[NH2]

and SEQ ID NO:S 31-34

TR-LSS YC SRSGHS CY

TR-LRQ YC RFQEEIKENTKNDKQ CY

TR-LTI YC TSYPD CI

TR-LED RYQEEC KENTK CDKQ.

Generally, a compound of the present invention is a cyclic peptide orpeptide analogue which is modified at its termini with hydrophobicmoieties. In certain embodiments, one or more amino acid residues withinthe peptide are substituted with other amino acid residues. Typically,such amino acid substitutions are conservative, i.e., the amino acidresidues are replaced with other amino acid residues having similarphysical and/or chemical properties. In embodiments wherein the compoundis a peptide analogues, the analogues is obtained by replacing at leastone amide linkage in the peptide with a substituted amide or an isostereof amide.

“Aromatic moiety:” refers to a moiety having an unsaturated cyclichydrocarbon group which has a conjugated (4n=2) π electron system,typical aromatic moieties include, but are not limited to, benzene,naphthalene, anthracene, azulene, indacene, and the like. In preferredembodiments, the aromatic moiety contains 5-20 carbons in the ringsystem, with 5-10 carbon atoms being particularly preferred.

“Substituted Aromatic Moiety:” refers to an aromatic moiety wherein oneor more hydrogen atoms are each independently replaced with othersubstituents.

“Heteroaromatic moiety:” refers to an aromatic moiety wherein one ormore of the ring carbon atoms is replaced with another atom such as N, Oor S. Typical heteroaromatic moieties include, but are not limited to,pyran, pyrazole, pyridine, pyrrolke, pyrazine, pyridazine, pyrimidine,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,selenophene, thiophere, tellurophene, xanthene and the like.

“Substituted Heteroaromatic moiety:” refers to a heteroaromatic moietywherein one or more hydrogen atoms are each independently replaced withother substituents.

Generally, a compound of the present invention is a cyclic peptide orpeptide analogue. The peptide or peptide analogue is modified at itstermini with hydrophobic moieties. In certain embodiments, one or moreamino acid residues within the peptide are substituted with other aminoacid residues. Typically, such amino acid substitutions areconservative, i.e., the amino acid residues are replaced with otheramino acid residues having physical and/or chemical properties similarto the residues they are replacing. Preferably, conservative amino acidsubstitutions are those wherein an amino acid is replaced with anotheramino acid encompassed within the same designated class, as will bedescribed more thoroughly below. In embodiments wherein the compound isa peptide analogue, the analogue is obtained by replacing at least oneamide linkage in the peptide with a substituted amide or an isostere ofamide.

The amino acid residues may be the genetically encoded L-amino acids,naturally occurring non-genetically encoded L-amino acids, syntheticL-amino acids or D-enantiomers of all of the above. The amino acidnotations used herein for the twenty genetically encoded L-amino acidsand common non-encoded amino acids are conventional and are as follows:

Amino Acid One-Letter Symbol Common Abbreviation Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val β-alanine bAla 2,3-diaminopropionic acid Dpr-α-aminoisobutyric acid Aib N-Methylglycine MeGly (sarcosine) OrnithineOrn Citrulline Cit t-buytlalanine t-Bua t-butylglycine t-BugN-methylisoleucine Melle phenylglycine Phg cyclohexylalanine ChaNorleucine Nle naphthylalanine Nal Pyridylananine 3-benzothienyl alanine4-chlorophenylalanine Phe(4-Cl) 2-fluorophenylalanine Phe(2-F)3-fluorophenylalanine Phe(3-F) 4-fluorophenylalanine 9 Penicillamine Pen1,2,3,4- Tic tetrahydroisoquinoline-3- carboxylic acid β-thienylalanineThi Methionine sulfoxide MSO Homoarginine hArg N-acetyl lysine AcLya2,4-diamino butyric acid Dbu p-aminophenylalanine Phe(pHN₂)N-methylvaline MeVal Homocysteine hCys Homoserine bSer ε-amino hexanoicacid Aha

The compounds that are encompassed within the scope of the invention arepartially defined in terms of amino acid residues of designated classes.The amino acids may be generally categorized into three main classes:hydrophilic amino acids, hydrophobic amino acids and Cysteine-like aminoacids, depending primarily on the characteristics of the amino acid sidechain. These amino classes may be further divided into subclasses.Hydrophilic amino acids include amino acids having acidic, basic orpolar side chains and hydrophobic amino acids including amino acidshaving aromatic or apolar side chains. Apolar amino acids ma be furthersubdivided to include, among others, aliphatic amino acids. Thedefinitions of the classes of amino acids as used herein are as follows:

Hydrophobic Amino Acid refers to an amino acid having a side chain thatis uncharged at physiological pH and that is repelled by aqueoussolution. Examples of genetically encoded hydrophobic amino acidsinclude Ile, Leu and Val. Examples of non-genetically encodedhydrophobic amino acids include t-BuA.

Aromatic Amino Acid refers to a hydrophobic amino acid having a sidechain containing at least one ring having a conjugated π-electron system(aromatic group). The aromatic group may be further substituted withsubstituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfanyl,nitro and amino groups, as well as others. Examples of geneticallyencoded aromatic amino acids include phenylalanine, tyrosine andtryptophan. Commonly encountered non-genetically encoded aromatic aminoacids include phenylglycine, 2-naphthylalanine, β-2-thienylalanine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,4-chloro-phenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and4-fluorophyenylalanine.

“Apolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is generally uncharged at physiological pH and that is notpolar. Examples of genetically encoded apolar amino acids includeglycine, proline and methionine. Examples of non-encoded apolar aminoacids include Cha.

“Aliphatic Amino Acid” refers to an apolar amino acid having a saturatedor unsaturated straight chain, branched or cyclic hydrocarbon sidechain. Examples of genetically encoded aliphatic amino acids includeAla, Leu, Val and Ile. Examples of non-encoded aliphatic amino acidsinclude Nle.

“Hydrophilic Amino Acid” refers to an amino acid having a side chainthat is attracted by aqueous solution. Examples of genetically encodedhydrophilic amino acids include Ser and Lys. Examples of non-encodedhydrophilic amino acids include Cit and hCys.

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Examples of genetically encoded acidic amino acids includeaspartic acid (aspartate) and glutamic acid (glutamate).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Examples of genetically encoded basic amino acidsinclude arginine, lysine and histidine. Examples of non-geneticallyencoded basic amino acids include the non-cyclic amino acids omithine,2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has a bond inwhich the pair of electrons shared in common by two atoms is held moreclosely by one of the atoms. Examples of genetically encoded polar aminoacids include asparagine and glutamine. Examples of non-geneticallyencoded polar amino acids include citrulline, N-acetyl lysine andmethionine sulfoxide.

“Cysteine-Like Amino Acid” refers to an amino acid having a side chaincapable of forming a covalent linkage with a side chain of another aminoacid residue, such as a disulfide linkage. Typically, cysteine-likeamino acids generally have a side chain containing at least one thiol(SH) group. Examples of genetically encoded cysteine-like amino acidsinclude cysteine. Examples of non-genetically encoded cysteine-likeamino acids include homocysteine and penicillamine.

As will be appreciated by those having skill in the art, the aboveclassification are not absolute—several amino acids exhibit more thanone characteristic property, and can therefore be included in more thanone category. For example, tyrosine has both an aromatic ring and apolar hydroxyl group. Thus, tyrosine has dual properties and can beincluded in both the aromatic and polar categories. Similarly, inaddition to being able to form disulfide linkages, cysteine also hasapolar character. Thus, while not strictly classified as a hydrophobicor apolar amino acid, in many instances cysteine can be used to conferhydrophobicity to a peptide.

Certain commonly encountered amino acids which are not geneticallyencoded of which the peptides and peptide analogues of the invention maybe composed include, but are not limited to, β-alanine (B-Ala) and otheromega-amino acids such as 3-aminopropionic acid (Dap),2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth;α-aminoisobutyric acid (Aib); ε-aminohyxanoic acid (Aha);δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly); omithine (Orn);citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG);N-methylisoleucine (MeIle); phenylglycine (phg); cyclohexylalanine(Cha); norleucine (Nle); 2-naphythylalanine (2-Nal);4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F));3-flurophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F));penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (MOS);homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid(Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH₂));N-methyl valine (MeVal); homocysteine (hCys) and homoserine (hSer).These amino acids also fall conveniently into the categories definedabove.

The classifications of the above-described genetically encoded andnon-encoded amino acids are summarized in Table 1, below. It is to beunderstood that Table 1 is for illustrative purposes only and does notpurport to be an exhaustive list of amino acid residues which maycomprise the peptides and peptide analogues described herein. Otheramino acid residues which are useful for making the peptides and peptideanalogues described herein can be found, e.g., in Fasman, 1989, CRCPractical Handbook of Biochemistry and Molecular Biology, CRC Press,Inc., and the references cited therein. Amino acids not specificallymentioned herein can be conveniently classified into the above-describedcategories on the basis of known behavior and/or their characteristicchemical and/or physical properties as compared with amino acidsspecifically identified.

TABLE 1 Classification Genetically Encoded Genetically Non-EncodedHydrophobic Aromatic F, Y, W Phg, Nal, Thi, Tic, Phe(4-Cl), Phe(2-F),Phe(3-F), Phe(4-F); Pyridyl Ala, Benzothienyl Ala Apolar M,G,P AliphaticA, V, L, I T-BuA, T-BuG, Melle, Nle, MeVal, Cha, bAla, MeGly, AibHydrophilic Acidic D, E Basic H, K, R Dpr, Orn, hArg, Phe(ρ-NH₂), DBU,A₂BU Polar Q, N, S, T, Y Cit, AcLys, MSO, hSer Cysteine-Like C Pen,hCys, B-methyl Cvs

A linking amino acid residue is one forming covalent linkages withanother so as to allow cyclization of the peptide. Examples of aminoacid residues which are capable of forming covalent linkages with oneanother include cysteine-like amino acids such as Cys, hCys, β-methylCys and Pen, which are capable of forming disulfide bridges with oneanother. Preferred cysteine-like amino acid residues include Cys andPen.

Amino acids used to cyclize a peptide need not be cysteine-like aminoacids. Pairs of amino acids that have side chain functional groupscapable of forming covalent linkages with one another can also be used.Such pairs of functional groups are known to those of skill in the artand include, inter alia, —COOH and —OH, —COOH and —NH₂, and —COOH and—SH. Thus, pairs of amino acids that can be used to cyclize a peptideinclude, inter alia, Asp and Lys; Glu and Lys; Asp and Arg; Glu and Arg;Asp and Ser; Glu and Ser; Asp and Thr; Glu and Thr; Asp and Cus; andGlu8 and Cys. Other pairs of amino acids which can be used to cyclizethe peptide will be apparent to those skilled in the art.

Equivalents of linking amino acids include groups used to cyclize apeptide such as any molecule having three functional groups—onefunctional group capable of forming a covalent linkage with a terminusof the peptide, a second functional group capable of forming a covalentlinkage with the second functional group of another group, and a thirdfunctional group capable of forming a covalent linkage with hydrophobicmoieties. Molecules having a suitable functional groups will be apparentto those skilled in the art. Examples of functional groups capable offorming a covalent linkage with the amino terminus of a peptide includecarboxylic acids and esters. Examples of functional groups capable offorming a covalent linkage with the carboxyl terminus of a peptideinclude —OH, —SH, —NH₂ and —NHR where R is (C₁-C₆) alkyl, (C₁-C₆)alkenyl and (C₁-C₆) alkynyl.

A variety of interlinkages useful to cyclize a peptide can be generatedby reaction between two functional groups suitable for forming suchinterlinkages, as well as reaction conditions suitable for forming suchinterlinkages, will be apparent to those of skill in the art.Preferably, the reaction conditions used to cyclize the peptides aresufficiently mild so as not to degrade or otherwise damage the peptide.Suitable groups for protecting the various functionalities as necessaryare well know in the art (see, e.g., Green & Wuts, 1991, 2^(nd) ed.,John Wiley & Sons NY), as are various reaction schemes for preparingsuch protected molecules.

The exocylic portions of the peptides represent a hydrophobic moiety.While not intending to be bound by any particular theory, it is believedthat when placed in aqueous solution, these hydrophobic moietiesinteract so as to confer the peptide with structural stability. Asignificant hydrophobic interaction for conferring structural stabilityis thought to be stacking of aromatic rings. Thus, in a preferredembodiment, R₁ and R₅ designate 1-5 amino acids, at least one of whichis an aromatic amino acid or an aromatic or heteroaromatic moiety. Morepreferably, each of R₁ and R₅ include an aromatic amino acid or anaromatic or heteroaromatic moiety. Suitable aromatic amino acids includeTyr and Phe being preferred. Suitable aromatic or heteroaromaticmoieties include phenyl, naphthyl, purine, pyrimidine, and the like.

In the peptides having the formula R₁—R₂—R₃—R₄—R₅, the symbol “—”between amino acid residues generally designates a backboneinterlinkage. Thus, the symbol “—” usually designates an amide linkage(—C(O)—NH). It is to be understood, however, that in all of the peptidesdescribed in the specific embodiments herein, one or more amide linkagesmay optionally be replaced with a linkage other than amide, preferably asubstituted amide or an isostere of an amide linkage. Thus, while thevarious R groups have generally been described in terms of amino acids,one having skill in the art will recognize that in embodiments havingnon-amide linkages, the term “amino acid” refers to other bifunctionalmoieties having side-chain groups similar to the side chains of theamino acids. For example, in embodiments having non-amide linkages, thephrase “acidic amino acid” refers to a bifunctional molecule capable offorming the desired backbone interlinkages and which has a side chaingroup similar to the side chain of an acidic amino acid. Substitutedamides generally include groups of the formula —C(O)—NR, where R is(C₁-C₆)alkyl, (C₁-C₆)alkenyl, (C₁-C₆)alkynyl, substituted (C₁-C₆)alkyl,substituted (C₁-C₆)alkenyl or substituted (C₁-C₆)alkynyl. Isosteres ofamide generally include, but are not limited to, —CH₂NH—, —CH₂S—,—CH₂CH2, —CH═CH— (cis and trans), —C(O)CH₂— and —CH₂S)—.

Compounds having such linkages and methods for preparing such compoundsare well-known in the art (see, e.g., Spatola, 1983, Vega Data 1(3) fora general review); Spatola, 1983, “Peptide Backbone Modifications” In:Chemistry and Biochemistry of Amino Acids Peptides and Proteins(Weinstein, ed.), Marcel Dekker, New York, p. 267 (general review);Morley, 1980, Trends Pharm. Sci. 1:463468; Hudson et al., 1979, Int. J.Prot. Res. 14:177-185 (—CH₂NH—, —CH₂CH₂—); Spatola et al., 1986, LifeSci. 38:1243-1249 (—CH₂—S); Hann, 1982, J. Chem. Soc. Perkin Trans. 1.1:307-314 (—CH═CH—, cis and trans); Jennings-White et al., Tetrahedron.Lett. 23:1392-1398 (—COCH₂—); European Patent Application EP 45665(1982) CA:97:39405 (—CH(OH)CH₂—); Holladay et al., 1983, TetrahedronLett. 24:4401-4404 (—C (OH)CH₂—); and Hruby, 1982, Life Sci. 31:189-199(—CH₂—S—).

In all of the aforementioned embodiments of the invention, it is to beunderstood that the phrase “amino acid” also refers to bifunctionalmoieties having amino acid-like side chains, as previously described.

Generally, active peptides or peptide analogues of the invention arethose that exhibit at least about 15% inhibition in the Trap assay setforth in the example. Preferably, active peptides of the invention oranalogues thereof will exhibit at least about 20% to 50% or even 80% ormore inhibition.

The peptides of the invention or analogues thereof, may be preparedusing virtually any art-known technique for the preparation of peptidesand peptide analogues. For example, the peptides may be prepared inlinear or non-cyclized form using conventional solution or solid phasepeptide syntheses and cyclized using standard chemistries. Preferably,the chemistry used to cyclize the peptide will be sufficiently mild soas to avoid substantially degrading the peptide. Suitable procedures forsynthesizing the peptides described herein as well as suitablechemistries for cyclizing the peptides are well known in the art.

Formation of disulfide linkages, if desired, is generally conducted inthe presence of mild oxidizing agents. Chemical, enzymatic or photolyticoxidation agents may be used. Various methods are known in the art,including those described, for example, by Tan, J. P. et al., 1979,Synthesis 955-957; Stewart et al., 1984, Solid Phase Peptide Synthesis.2d Ed., Pierce Chemical Company Rockford, Ill.; Ahmed et al., 1975, J.Biol. Chem. 250:8477-8482; and Pennington et al., 1991 Peptides 1990164-166, Giralt and Andreu, Eds., ESCOM; Leiden, The Netherlands. Anadditional alternative is described by Kamber et al., 1980, Helv ChimActa, 63:899-915. A method conducted on solid supports is described byAlbericio, 1985, Int. J. Peptide Protein Res., 26:92-97. Any of thesemethods may be used to form disulfide linkages in the peptides of theinvention. Preferred methods for effecting disulfide-bridge formationfor the peptides described herein are provided in the examples.

If the peptide is composed entirely of gene-encoded amino acids, or aportion of it is so composed, the peptide or the relevant portion mayalso be synthesized using conventional recombinant genetic engineeringtechniques. The isolated peptides, or segments thereof, are thencondensed, and oxidized, as previously described, to yield a cyclicpeptide.

For recombinant production, a polynucleotide sequence encoding a linearform of the peptide is inserted into an appropriate expression vehicle,i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence, or in thecase of an RNA viral vector, the necessary elements for replication andtranslation. The expression vehicle is then transfected into a suitabletarget cell which will express the linear form of the cyclic peptide.Depending on the expression system used, the expressed peptide is thenisolated by procedures well-established in the art. Methods forrecombinant protein and peptide production are well known in the art(see, e.g., Maniatis et al., 1989, Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, N.Y.).

A variety of host-expression vector systems may be utilized to expressthe peptides described herein. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or robacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems.

The expression elements of the expression systems vary in their strengthand specificities. Depending on the host/vector system utilized, any ofa number of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of baceriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedron promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genomes of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5 K promoter) may beused; when generating cell lines that contain multiple copies ofexpression product, SV40-, BPV- and EBV-based vectors may be used withan appropriate selectable marker.

In cases where plant expression vectors are used, the expression ofsequences encoding the peptides of the invention may be driven by any ofa number of promoters. For example, viral promoters such as the 35S RNAand 19S RNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987,EMBO J., 6:307-311) may be used; alternatively, plant promoters such asthe small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680;Broglie et al., 1984, Science 224:838-843) or heat shock promoters,e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell.Biol. 6:599-565) may be used. These constructs can be introduced intoplant cells using Ti plasmids, Ri plasmids, plant virus vectors, directDNA transformation, microinjection, electroporation, etc. For reviews ofsuch techniques see, e.g., Weissbach & Weissbach, 1988, Methods forPlant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463;and Grierson & Corey, 1988, Plant Molecular Biology, 2^(nd) Ed.,Blackie, London, Ch. 7-9.

In one insect expression system that may be used to produce the peptidesof the invention, Autographa californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express the foreign genes. The virusgrows in Spodoptera frugiperda cells. A coding sequence may be clonedinto non-essential regions (for example the polyhedron gene) of thevirus and placed under control of an AcNPV promoter (for example, thepolyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (e.g., see Smith et al., 1983, J. Virol., 46:584;Smith, U.S. Pat. No. 4,215,051). Further examples of this expressionsystem may be found in Current Protocols in Molecular Biology, Vol. 2,Ausubel et al., eds., Greene Publish. Assoc. & Wiley Interscience.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g. Region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promotermay be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci.USA, 79:7415-7419; Mackett et al., 1984, J. Virol., 49:857-864; Panicaliet al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).

Other expression systems for producing linear or non-cyclized forms ofthe cyclic peptides of the invention will be apparent to those havingskill in the art.

The peptides and peptide analogues of the invention can be purified byart-known techniques such as high performance liquid chromatography, ionexchange chromatography, gel electrophoresis, affinity chromatographyand the like. The actual conditions used to purify a particular peptideor analogue will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, etc., and will be apparent to thosehaving skill in the art.

For affinity chromatography purification, any antibody whichspecifically binds the peptides or peptide analogues may be used. Forthe production of antibodies, various host animals, including but notlimited to rabbits, mice, rats, etc., may be immunized by injection witha linear or cyclic peptide. The peptide may be attached to a suitablecarrier, such as BSA, by means of a side chain functional group orlinkers attached to a side chain functional group. Various adjuvants maybe used to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, sufrace activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacilli Calmette-Duerin) andCorynebacterium parvum.

Monoclonal antibodies to a peptide may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include but are not limited to thehybridoma technique originally described by Koehler and Milstein, 1975,Nature, 256:495-497; the human B-cell hybridoma technique, Kosbor etal., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc. Natl. Acad.Sci., USA, 80:2026-2030 and tghe EBV-hybridoma technique (Cole et al.,1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96 (1985)). In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,USA, 81: 6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takadaet al., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to producecyclic peptide-specific single chain antibodies.

Antibody fragments which contain deletions of specific binding sites maybe generated by known techniques. For example, such fragments includebut are not limited to F(ab′)₂ fragments, which can be produced bypepsin digestion of the antibody molecule and Fab fragments, which canbe generated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science 246: 1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for the cyclicpeptide of interest

The antibody or antibody fragment specific for the desired cyclicpeptide can be attached, for example, to agarose, and theantibody-agarose complex is used in immunochromatography to purifycyclic peptides of the invention. See, Scopes, 1984, ProteinPurification: Principles and Practice, Spriger-Verlag New York, Inc.,N.Y., Livingstone, 1974, Methods Enzymology: ImmunoaffinityChromatography of Proteins 34:723-731.

The compounds of the invention, may be administered to a subject per seor in the form of a pharmaceutical composition. Pharmaceuticalcompositions comprising the compounds of the invention may bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. Pharmaceutical compositions may be formulated inconventional manner using one or more physiological acceptable carriers,diluents, excipients or auxiliaries which facilitate processing of theactive peptides or peptide analogues into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. For topical administration the compounds of theinvention may be formulated as solutions, gels, ointments, creams,suspensions, etc. as are well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintrapertoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Alternatively, the compounds may be in powder form for constitution witha suitable vehicle, e.g., sterile pyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be readily formulated bycombining the active peptides or peptide analogues with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thecompounds of the invention to be formulated as tablets, pills, dragees,capsules, liquids gels, syrups, slurries, suspensions and the like, fororal ingestion by a patient to be treated. For oral solid formulationssuch as, for example, powders, capsules and tablets, suitable excipientsinclude fillers such as sugars, such as lactose, sucrose, mannitol andsorbitol; cellulose preparations such as maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium, carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP); granulating agents; and binding agents. Ifdesired, disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, atgar, or alginic acid or a salt thereof such assodium alginate.

If desired, solid dosage forms may be sugar-coated or enteric-coatedusing standard techniques.

For oral preparations such as, for example, suspensions, elixirs andsolutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

For buccal administration, the compounds may take the form of tablets,lozenges, etc. formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or my intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat may be used to deliver peptides and peptide analogues of theinvention. Certain organic solvents such as dimethylsulfoxide also maybe employed, although usually at the cost of greater toxicity.Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

As the compounds of the invention may contain charged side chains ortermini, they may be included in any of the above-described formulationsas the free acids or bases or as pharmaceutically acceptable salts.Pharmaceutically acceptable salts are those salts which substantiallyretain the antimicrobial activity of the free bases and which areprepared by reaction with inorganic acids. Pharmaceutical salts tend tobe more soluble in aqueous and other protic solvents than are thecorresponding free base forms.

The compounds of the invention will generally be used in an amounteffective to achieve the intended purpose. For use to preventosteoclastogenesis and/or osteoclast activity, the compounds of theinvention, or pharmaceutical compositions thereof, are administered orapplied in a therapeutically effective amount. By therapeuticallyeffective amount is meant an amount effective ameliorate or prevent thesymptoms, or prolong the survival of, the patient being treated.Determination of a therapeutically effective amount is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the compounds which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5to 1 mg/kg/day. Therapeutically effective serum levels may be achievedby administering multiple doses each day.

In cases of local administration or selective uptake, the effectivelocal concentration of the compounds may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

The amount of compound administered will, of course, be dependent on thesubject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The therapy may be repeated intermittently while symptoms detectable oreven when they are not detectable. The therapy may be provided alone orin combination with other drugs.

Preferably, a therapeutically effective dose of the compounds describedherein will provide therapeutic benefit without causing substantialtoxicity.

Toxicity of the compounds described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD5₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Compoundswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulation a dosage range that is not toxic for use in human. Thedosage of the compounds described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or not toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (See,e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics,Ch.1, p.1).

The invention having been described, the following examples are offeredby way of illustration and not limitation.

EXAMPLES Example 1

Recently, therapeutic peptidomimetics that interfere with the TNF/TNFreceptor (1) interaction have been developed based on atomic structuresdeduced from the crystal structures of TNF-α and the TNFβ/TNF receptor(1) complex (Takasaki et al. Nature Biotechnology, 15:1266-1270, 1997).The most critical TNF-α recognition site was localized to the first loopof the third domain of TNF receptor (1) (residues 107-114). Apeptidomimetic (WP9QY) engineered to mimic this recognition siteefficiently antagonized the effects of TNF-binding to the TNF-α receptor(1) in L929 lymphocytes.

This peptide (5-500 μM) was tested for its effect on osteoclastformation using the co-culture system induced by 1,250H2D3 and PGE2.Osteoclastogenesis was dose-and time-dependently inhibited by thepeptide (IC₅₀=250 μM) but the IC₅₀ was 50-fold higher than what wasrequired for the TNF/TNF receptor (1) interaction (5 μM). Thisdifference suggests that the peptide inhibits osteoclastogenesis byinterfering not with the TNF/TNF receptor (1) interaction but withanother related ligand-receptor pair such as TRANCE/RANK. This wasconfirmed by demonstrating that WP9QY inhibits TRANCE-induced marrowcultures. There was a reciprocal dose-dependence of WP9QY and TRANCE.Thus, WP9QY is capable of interfering not only with TNF/TNF receptor (1)interaction but also with RANK Ligand/RANK interaction, therebydecreasing the osteoclastogenic potential of this cytokine.

TRANCE/RANK inhibitors of the invention may be evaluated for theirability to inhibit osteoclastogenesis and osteoclast function using theassay described herein.

Example 2

Identification of Osteoclasts Formed in Vitro

TRAP refers to tartrate resistant acid phosphatase which identifiesosteoclast like cells.

Osteoprotegerin (OPG) is a naturally occurring secreted protein withhomology to members of the TNF receptor family. Administration of OPG invivo inhibits osteoclastogenesis and associated bone resorption andblocks the pathological increase in osteoclast numbers and activity seenin animal models that mimic osteopenic disorders in humans. OPG can beused as a positive control in the TRAP assay.

Cytochemical staining for TRAP is widely used for identifyingosteoclasts in vivo and in vitro. Naphthol AS-MX phosphate 5 mg. Sigma,St. Louis, Mo.) is resolved in 0.5 ml of N,N-dimethylformamide (Wako).Thirty milligrams of fast red violet LB salt (Sigma) and 50 ml of 0.1 Msodium acetate buffer (pH 5.0) containing 50 mM sodium tartratet areadded to the mixture (the TRAP-staining solution). Cells are fixed with3.7% (v/v) formaldehyde in Ca²⁻- and Mg²⁺-free phosphate-buffered saline[PBS(−)] for 10 min. fixed again with ethanol-acetone (50:50, v:v) for 1min. and incubated with the TRAP-staining solution for 10 min. at roomtemperature. TRAP-positive osteoclasts appear as red cells. Theincubation period longer than 10 min. should be avoided since cellsother than osteoclasts become weakly positive with time. After staining,cells are washed with distilled water, and TRAP-positive multinucleatedcells having three or more nuclei are counted as osteoclasts under amicroscope. (G. C. Nicholson, J. M. Mosely, P. M. Sexton, F. A. O.Mendelssohn, and T. J. Martin, J. Clin. Invest. 78, 355 (1986), which isincorporated herein by reference).

34 1 5 PRT Artificial Sequence Description of Artificial Sequence NovelSequence 1 Asp Arg Gly Trp Ala 1 5 2 6 PRT Artificial SequenceDescription of Artificial Sequence Novel Sequence 2 Asp Gly Asp Leu AlaThr 1 5 3 6 PRT Artificial Sequence Description of Artificial SequenceNovel Sequence 3 Ser Asp Phe Ala Thr Glu 1 5 4 12 PRT ArtificialSequence Description of Artificial Sequence Novel Sequence 4 Val Thr LysThr Ser Ile Lys Ile Pro Ser Ser His 1 5 10 5 11 PRT Artificial SequenceDescription of Artificial Sequence Novel Sequence 5 Thr Lys Thr Ser IleLys Ile Pro Ser Ser His 1 5 10 6 10 PRT Artificial Sequence Descriptionof Artificial Sequence Novel Sequence 6 Lys Thr Ser Ile Lys Ile Pro SerSer His 1 5 10 7 7 PRT Artificial Sequence Description of ArtificialSequence Novel Sequence 7 Tyr Trp Ser Asn Ser Glu Phe 1 5 8 5 PRTArtificial Sequence Description of Artificial Sequence Novel Sequence 8Tyr Trp Asn Ser Glu 1 5 9 6 PRT Artificial Sequence Description ofArtificial Sequence Novel Sequence 9 Pro Asp Gln Asp Ala Pro 1 5 10 5PRT Artificial Sequence Description of Artificial Sequence NovelSequence 10 Pro Asp Ser Trp His 1 5 11 4 PRT Artificial SequenceDescription of Artificial Sequence Novel Sequence 11 Ser Lys Glu Leu 112 4 PRT Artificial Sequence Description of Artificial Sequence NovelSequence 12 Glu Ile Glu Phe 1 13 6 PRT Artificial Sequence Descriptionof Artificial Sequence Novel Sequence 13 Ser Arg Ser Gly His Ser 1 5 1415 PRT Artificial Sequence Description of Artificial Sequence NovelSequence 14 Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln1 5 10 15 15 5 PRT Artificial Sequence Description of ArtificialSequence Novel Sequence 15 Thr Ser Tyr Pro Asp 1 5 16 5 PRT ArtificialSequence Description of Artificial Sequence Novel Sequence 16 Lys GluAsn Thr Lys 1 5 17 5 PRT Artificial Sequence Description of ArtificialSequence Novel Sequence 17 Arg Tyr Gln Glu Glu 1 5 18 5 PRT ArtificialSequence Description of Artificial Sequence Novel Sequence 18 Tyr ValLys Gln Glu 1 5 19 4 PRT Artificial Sequence Description of ArtificialSequence Novel Sequence 19 Tyr Lys His Arg 1 20 9 PRT ArtificialSequence Description of Artificial Sequence Novel Sequence 20 Tyr CysAsp Arg Gly Trp Ala Cys Tyr 1 5 21 10 PRT Artificial SequenceDescription of Artificial Sequence Novel Sequence 21 Tyr Cys Asp Gly AspLeu Ala Thr Cys Tyr 1 5 10 22 10 PRT Artificial Sequence Description ofArtificial Sequence Novel Sequence 22 Tyr Cys Ser Asp Phe Ala Thr GluCys Tyr 1 5 10 23 16 PRT Artificial Sequence Description of ArtificialSequence Novel Sequence 23 Tyr Cys Val Thr Lys Thr Ser Ile Lys Ile ProSer Ser His Cys Tyr 1 5 10 15 24 14 PRT Artificial Sequence Descriptionof Artificial Sequence Novel Sequence 24 Tyr Cys Lys Thr Ser Ile Lys IlePro Ser Ser His Cys Tyr 1 5 10 25 11 PRT Artificial Sequence Descriptionof Artificial Sequence Novel Sequence 25 Tyr Cys Tyr Trp Ser Asn Ser GluPhe Cys Tyr 1 5 10 26 8 PRT Artificial Sequence Description ofArtificial Sequence Novel Sequence 26 Cys Tyr Trp Asn Ser Glu Cys Tyr 15 27 10 PRT Artificial Sequence Description of Artificial Sequence NovelSequence 27 Tyr Cys Pro Asp Gln Asp Ala Pro Cys Tyr 1 5 10 28 11 PRTArtificial Sequence Description of Artificial Sequence Novel Sequence 28Tyr Cys Pro Asp Ser Trp His Cys Tyr Asp Glu 1 5 10 29 12 PRT ArtificialSequence Description of Artificial Sequence Novel Sequence 29 Tyr CysSer Lys Glu Leu Cys Tyr Val Lys Gln Glu 1 5 10 30 11 PRT ArtificialSequence Description of Artificial Sequence Novel Sequence 30 Tyr CysGlu Ile Glu Phe Cys Tyr Lys His Arg 1 5 10 31 10 PRT Artificial SequenceDescription of Artificial Sequence Novel Sequence 31 Tyr Cys Ser Arg SerGly His Ser Cys Tyr 1 5 10 32 19 PRT Artificial Sequence Description ofArtificial Sequence Novel Sequence 32 Tyr Cys Arg Phe Gln Glu Glu IleLys Glu Asn Thr Lys Asn Asp Lys 1 5 10 15 Gln Cys Tyr 33 9 PRTArtificial Sequence Description of Artificial Sequence Novel Sequence 33Tyr Cys Thr Ser Tyr Pro Asp Cys Ile 1 5 34 15 PRT Artificial SequenceDescription of Artificial Sequence Novel Sequence 34 Arg Tyr Gln Glu GluCys Lys Glu Asn Thr Lys Cys Asp Lys Gln 1 5 10 15

What is claimed is:
 1. A method for treating bone loss in a patient,which method comprises the step of administering to said patient anamount of an inhibitor of TNF-related activator induced cytokine(TRANCE)/Receptor activator of NF-B ligand (RANK) that is effective toinhibit osteoclastogenesis or osteoclast function, wherein the inhibitoris a specific peptide having the formula: R1—R2—R3—R4—R5 wherein: R1 is1-5 amino acid residues; R2 is a linking amino acid residue; R3 isselected from the group consisting of DRGWA (SEQ ID NO:1); DGDLAT (SEQID NO:2); SDFATE (SEQ ID NO:3); VKTSIKIPSSH (SEQ ID NO:4); TKTSIKIPSSH(SEQ ID NO:5); KTSIKIPSSH (SEQ ID NO:6); YWSNSEF (SEQ ID NO:7); YWNSE(SEQ ID NO:8); PDQDAP (SEQ ID NO:9); PDSWH (SEQ ID NO:10); SKEL (SEQ IDNO:11); EIEF (SEQ ID NO:12); SRSGHS (SEQ ID NO:13); RFQEEIKENTKNDKQ (SEQID NO:14); TSYPD (SEQ ID NO:15); KENTK (SEQ ID NO:16); and SEQ IDNOS:1-16 with one or more amino acid residues substituted with anotherresidue in the same designated class; R4 is a linking amino acidresidue; R5 is 1-5 amino acid residues; and wherein R2 and R4 are boundto each other, thereby forming a cyclic portion which includes R2, R3and R4 with R1 and R5 forming exocyclic portions, and one or both of R1and R5 comprising at least one tyrosine or phenylalanine.
 2. The methodaccording to claim 1 wherein: R1 is Tyrosine (Y); R2 is Cysteine (C); R3comprises the amino acid sequence EIEF (SEQ ID NO:12); R4 is Cysteine(C); and R5 is Tyrosine (Y).
 3. A method for treating bone loss in apatient, which method comprises the step of administering to saidpatient an amount of an inhibitor of TNF-related activator inducedcytokine (TRANCE)/Receptor activator of NF-B ligand (RANK) that iseffective to inhibit osteoclastogenesis or osteoclast function, whereinthe inhibitor is a cyclic peptide selected from the group consisting of:YC DRGWA CY (SEQ ID NO:20); YC DGDLAT CY (SEQ ID NO:21); YC SDFATE CY(SEQ ID NO:22); YC VTKTSIKIPSSH CY (SEQ ID NO:23); YC KTSIKIPSSH CY (SEQID NO:24; YC YWSNSEF CY (SEQ ID NO:25); C YWNSE CY (SEQ ID NO:26); YCPDQDAP CY (SEQ ID NO:27); YC PDSWH CYDE (SEQ ID NO:28); YC SKEL CYVKQE(SEQ ID NO:29); YC EIEF CYKHR (SEQ ID NO:30); TR-LSS YC SRSGHS CY (SEQID NO;31); TR-LRQ YC RFQEEIKENTKNDKQ CY (SEQ ID NO;32); TR-LTI YC TSYPDCI (SEQ ID NO;33); and TR-LED RYQEEC KENTK CDKQ. (SEQ ID NO:34).


4. The method of claim 3 wherein the inhibitor is selected from thegroup consisting of SEQ ID NOS:20-30 with amidated C termini.
 5. Themethod according to claim 3 wherein the inhibitor is selected from thegroup consisting of: TR-LSS YC SRSGHS CY (SEQ ID NO;31); TR-LRQ YCRFQEEIKENTKNDKQ CY (SEQ ID NO;32); TR-LTI YC TSYPD CI (SEQ ID NO;33);and TR-LED RYQEEC KENTK CDKQ. (SEQ ID NO:34).


6. The method according to claim 3 wherein the inhibitor is selectedfrom the group consisting of: YC VTKTSIKIPSSH CY (SEQ ID NO:23); and YCKTSIKIPSSH CY (SEQ ID NO:24).


7. The method according to claim 6 wherein the C-terminal of theinhibitor is amidated.